Reducing non-target nucleic acid dependent amplifications: amplifying repetitive nucleic acid sequences

ABSTRACT

The present invention provides for compositions and methods for amplifying target nucleic acids using nucleic acid primers designed to limit non-target nucleic acid dependent priming events. The present invention permits amplifying and quantitating the number of repetitive units in a repetitive region, such as the number of telomere repetitive units.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application claims benefit of U.S. patent application Ser. No.10/355,626 filed Jan. 31, 2003 which claims priority to U.S. ProvisionalPatent Application No. 60/353,591 filed Jan. 31, 2002, the entirecontents of which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Number K01AG 00767 awarded by the National Institutes of Health. The Governmenthas certain rights to this invention.

FIELD OF THE INVENTION

The invention relates to the field of recombinant DNA technology.Specifically, the present invention is drawn to compositions and methodsfor amplifying target nucleic acids, especially direct amplification ofrepetitive nucleic acid sequences.

BACKGROUND OF THE INVENTION

Detecting the presence of target nucleic acids is vital in numerousapplications in medical diagnostics, forensics, genetic analysis, andpublic health. For instance, identifying specific DNA sequences iscritical for diagnosing inherited disorders, determining susceptibilityto disease, and identifying causal agents of infectious diseases.Polymerase chain reaction (PCR) provides a highly sensitive method fordetecting the presence of target nucleic acids by selectiveamplification of target nucleic acids. The method relies on use ofoligonucleotide primers that hybridize to opposite ends of a targetnucleic acid segment, an amplicon, and prime copying of the nucleic acidsegment by a polymerase. Reiterative rounds of DNA synthesis,denaturation, and reannealing allows exponential amplification of agiven target nucleic.

Primer selection is a major determinant in the success or failure of theamplification reaction. Critical factors include primer length, meltingtemperature (T_(m)), sequence specificity, complementary primersequences, G/C content, and 3′-terminal region sequence. In general,primers must hybridize with specificity to the target nucleic acid butnot hybridize to and amplify non-target nucleic acid sequences.

Amplifying non-target nucleic acids becomes problematic when the 3′ endof a primer is complementary to another primer. These primers will tendto hybridize to each other, which are then extended by polymerase toform “primer-dimer” products.

Subsequent amplification of primer-dimers leads to depletion of primers,resulting in reduced sensitivity or even failure to amplify the intendedtarget nucleic acid. Performing primer extensions or preamplificationannealing at temperatures limiting primer-primer hybrids (e.g., “hotstart” PCR, D'Aquila, R. T. et al., Nucleic Acids Res. 19: 3749 (1991);“touchdown” PCR, Don, R. H. et al., Nucleic Acids Res. 19: 4008 (1991))or adjusting buffer components to increase hybridization stringency mayminimize primer-dimer interference. However, the presence of excessprimers during PCR reactions allows even weak complementarity at the 3′terminal region to generate these interfering side products.

Although choosing different regions of the target nucleic acid forselecting primers provides a basis for limiting non-target nucleic aciddependent amplifications, constraints on selecting sequences forgenerating primers can limit the choice of alternative primer designs.For example, directly amplifying short, tandem repetitive sequences suchas telomere repeats is difficult since the primers for these sequenceswill always have some degree of complementarity. These repetitivesequences do not normally afford a choice in primer sequences forlimiting formation of primer-dimer products. Consequently, currentmethods for estimating telomere lengths rely on restriction enzymedigestion of genomic DNA followed by hybridization with repeat sequences(terminal restriction fragment analysis; see Harley, C. B. et al, Nature345: 458-460 (1990)), indirect amplification of repeats using uniquesequences positioned outside of the repeat region (see Kozlowski, M. R.et al., U.S. Pat. No. 5,741,677), fluorescence in situ hybridization(see Henderson, S. J. Cell Biol. 134: 1-12 (1996)), or flow cytometrymethods (see Hultdin, M. Nucleic Acids Res. 26: 3651-3656 (1998)).Generally, these procedures are time consuming or require substantialquantities of DNA. Since the copy number of telomere repeats and othertandemly repetitive sequences in a cell correlate with the physiologicalor diseased states of a cell, there is a need for compositions andmethods for rapidly amplifying and quantitating these sequences whilegenerally avoiding competing primer-dimer side reactions duringamplification.

SUMMARY OF THE INVENTION

In accordance with the objects outlined above, the present inventionprovides methods for amplifying target nucleic acids while limitingnon-target nucleic acid dependent amplifications. In a preferredembodiment, the method comprises contacting a target nucleic acidcomprising substantially complementary first and second strands with afirst and second primer, wherein the first primer hybridizes to thefirst strand and the second primer hybridizes to the second strand ofthe target nucleic acid, and wherein the hybridized primers are capableof primer extension when hybridized to their respective strands. Atleast one nucleotide of the first primer is altered to produce amismatch between altered residue and the 3′ terminal nucleotide residueof the second primer when first and second primers hybridize to eachother. The target nucleic acid is subsequently amplified by polymerasechain reaction.

In another aspect, the method for amplifying the target nucleic acidfurther comprises altering at least one nucleotide residue of the secondprimer to produce a mismatch between the altered residue on the secondprimer and the 3′ terminal nucleotide residue of the first primer whenthe primers hybridize to each other.

In another preferred embodiment, the present invention provides formethods of amplifying repetitive units in a repetitive region of atarget nucleic acid. In one embodiment, the method comprises contactinga target nucleic acid comprising substantially complementary first andsecond strands with a first and second primer, wherein the first primerhybridizes to at least one repetitive unit of the first strand and thesecond primer hybridizes to at least one repetitive unit of the secondstrand of the target nucleic acid, and wherein the hybridized primersare capable of primer extension when hybridized to their respectivestrands. At least one nucleotide residue of the first primer is alteredto a produce a mismatch between the altered residue and a nucleotideresidue of at least one repetitive unit of the first strand, wherein thealtered residue also produces a mismatch with the 3′ terminal nucleotideof the second primer when first and second primers hybridize to eachother. Target nucleic acids are subsequently amplified by polymerasechain reaction.

In another aspect, the method for amplifying repetitive sequencesfurther comprises altering at least one nucleotide residue of the secondprimer to produce a mismatch between the altered residue and anucleotide residue of at least one repetitive unit of the second strand,wherein the altered residue of the second primer also produces amismatch with the 3′ terminal nucleotide of the first primer when theprimers hybridize to each other.

In another preferred embodiment for amplifying repetitive units in arepetitive region, the method comprises contacting the substantiallycomplementary first and second strands with a first and second primer,wherein the first primer hybridizes to more than one repetitive unit ofthe first strand and the second primer hybridizes to more than onerepetitive unit of the second strand of the target nucleic acid, andwherein the hybridized primers are capable of primer extension whenhybridized to their respective strands. Nucleotide residues of the firstprimer are altered to produce mismatches between the altered residuesand nucleotide residues at the identical nucleotide position of eachrepetitive unit of the first strand, wherein the altered residues alsoproduce a mismatch with the 3′ terminal nucleotide residue of the secondprimer when the first and second primers hybridize to each other. Thetarget nucleic acids are then amplified.

In another aspect, the method for amplifying repetitive sequencesfurther comprises altering the nucleotide residues of the second primerto produce mismatches between the altered residues and the nucleotideresidue at the identical position of each repetitive unit of the secondstrand, wherein the altered residues of the second primer also producemismatches with the 3′ terminal nucleotide residue of the first primerwhen the primers hybridize to each other.

In a preferred embodiment, the present invention provides methods fordetermining the number of repetitive units in a repetitive region of atarget nucleic acid, such as telomeres, by measuring the amount ofamplified target nucleic acids. These methods find applications incancer diagnosis and examination of cell senescence.

In accordance with the described methods, the present invention furtherprovides compositions for amplifying the subject target nucleic acids,including telomere repetitive regions.

DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B depict the sequence of the oligonucleotide primer pairs,tel 1 (SEQ ID NO: 1) and tel 2 (SEQ ID NO: 2), used to amplify humantelomeric repetitive units (SEQ ID NOS: 3, 4). Shown are thehybridization schemes of the primers to the telomere repetitivesequences and hybridization of the primers to each other. The tel 1primer can hybridize to any available complementary 31 basepair stretchalong the strand of telomeric DNA oriented 5′ to 3′ toward thecentromere. The tel 2 primer can hybridize to any complementary 33basepair stretch along the strand oriented 5′ to 3′ toward the end ofthe chromosome. For each primer, nucleotide residues are altered toproduce mismatches between the altered residue and nucleotide residuesat the identical nucleotide position of each repetitive unit to whichthe primer hybridizes. Thus, for tel 1 and tel 2, every sixth base ismismatched. To limit primer-dimer products, the altered residues of eachprimer also produce a mismatch with the 3′ terminal nucleotide residueof the other primer when the primers hybridize to each other, thusblocking extension by polymerase. In addition, the 5′ terminal regionsof the primer are designed so as to not basepair with the telomericrepeats. These noncomplementary 5′ terminal region sequences prevent the3′ ends of the PCR products from initiating DNA synthesis in the middleof telomere amplification products. By designing the telomere specificprimers to have similar GC content, and hence similar T_(m)s (seeExample 1), undesirable amplifications are further reduced.

FIG. 2 shows standard curves used to measure relative T/S ratios, wherethe T/S ratio is the telomere (T) to single copy gene (S) ratio (seeExample 2). Five DNA concentrations over an eight-fold range weregenerated by serial dilution (dilution factor .about.1.68) and aliquotedinto microtiter plate wells; the final amounts per well ranged from12.64 ng to 100 ng, with the middle quantity approximately matching thatof the samples being assayed. The C_(t) of a DNA sample is thefractional number of PCR cycles to which the sample must be subjected inorder to accumulate enough product to cross a set threshold of magnitudeof fluorescent signal. Any individual or pooled human DNA sample may beused to create the standard curves, as long as each assayed sample'sC_(t) falls within the range of Cts of the standard curves (O=singlecopy gene 36B4; Δ telomere)

FIG. 3 shows correlation of relative T/S ratios determined by real timequantitative PCR using the primers described herein and the meantelomere restriction fragment (TRF) lengths determined by traditionalSouthern hybridization analysis. The DNA samples used for the analysiswere from blood drawn from 21 individuals. All relative T/S ratiosplotted have values .gtoreq.1.0 because the initial T/S ratiosdetermined using the standard curves have all been normalized to thelowest T/S ratio (0.69) observed among the samples. The equation for thelinear regression line best fitting the data is shown.

FIG. 4 shows amplification products separated by agarose gelelectrophoresis and visualized by ethidium bromide staining and UVillumination. After 25 cycles of PCR, amplified human telomere DNAappears as a smear with the highest intensity beginning at about 76basepairs and progressively fading to background around 400 basepairs(human genomic DNA). A smear of products beginning from about 76basepairs is also obtained when a target nucleic acid templatecontaining a known number of human telomere repetitive units, (TTAGGG)₂₀(SEQ ID NO: 5), is used in the amplification. When human genomic DNA isomitted (i.e., buffer) or replaced with E. coli. DNA, no product isdetectable up to 25 PCR cycles. DNA length standards are shown (123 byladder).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions useful inamplifying target nucleic acids while limiting non-target nucleic aciddependent amplification reactions. Specifically, the present inventionprovides for primers that limit amplification of primers hybridized toeach other while allowing amplification of the target nucleic acid.Generally, the primers of the present invention comprise altered ormutated nucleotide residues such that when the primers hybridize to eachother, the 3′ terminal residue of one primer forms a mismatch with thealtered or mutated nucleotide residue on the other primer, thuspreventing primer extension. The present invention finds use in reducingPCR background and for amplifying and quantitating repetitive sequences,especially telomere repetitive sequences.

By “primer”, “primer nucleic acid”, “oligonucleotide primer” orgrammatical equivalents as used herein is meant a nucleic acid that willhybridize to some portion of the target nucleic acid. The primers of thepresent invention are designed to be substantially complementary to atarget sequence such that hybridization of the target sequence and theprimers of the present invention occurs, and proper 3′ base pairingallows primer extension to take place. Such complementarity need not beperfect. Thus, by “complementary” or “substantially complementary”herein is meant that the probes are sufficiently complementary to thetarget sequences to hybridize under normal reaction conditions.Deviations from perfect complementary are permissible so long asdeviations are not sufficient to completely preclude hybridization.However, if the number of alterations or mutations is sufficient suchthat no hybridization can occur under the least stringent ofhybridization conditions, as defined below, the sequence is not acomplementary target sequence.

The primers of the present invention comprise nucleic acids. By “nucleicacid” or “oligonucleotide” or grammatical equivalents herein is meant atleast two nucleotides covalently linked together. A nucleic acid of thepresent invention will generally contain phosphodiester bonds, althoughin some cases, nucleic acid analogs are included that may have alternatebackbones, comprising, for example, phosphoramide (Beaucage, S. L. etal., Tetrahedron 49: 1925-1963 (1993) and references therein; Letsinger,R. L. et al., J. Org. Chem. 35: 3800-3803 (1970); Sprinzl, M. et al.,Eur. J. Biochem. 81: 579-589 (1977); Letsinger, R. L. et al., NucleicAcids Res. 14: 3487-3499 (1986); Sawai et al., Chem. Lett. 805 (1984);Letsinger, R. L. et al., J. Am. Chem. Soc. 110: 4470 (1988); and Pauwelset al., Chemica Scripta 26:141-149 (1986)), phosphorothioate (Mag, M. etal., Nucleic Acids Res. 19: 1437-1441 (199); and U.S. Pat. No.5,644,048), phosphorodithioate (Briu et al., J. Am. Chem. Soc. 111: 2321(1989)), O-methylphosphoroamidite linkages (see Eckstein, F.,Oligonucleotides and Analogues: A Practical Approach, Oxford UniversityPress, 1991), and peptide nucleic acid backbones and linkages (Egholm,M. Am. Chem. Soc. 114: 1895-1897 (1992); Meier et al., Chem. Int. Ed.Engl. 31: 1008 (1992); Egholm, M Nature 365: 566-568 (1993); Carlsson,C. et al. Nature 380: 207 (1996), all of which are incorporated byreference). Other analog nucleic acids include those with positivebackbones (Dempcy, R. O. et al., Proc. Natl. Acad. Sci. USA 92:6097-6101 (1995)); non-ionic backbones (U.S. Pat. Nos. 5,386,023;5,637,684; 5,602,240; 5,216,141; and 4,469,863; Kiedrowshi et al.,Angew. Chem. Intl. (Ed. English) 30: 423 (1991); Letsinger, R. L. etal., J. Am. Chem. Soc. 110: 4470 (1988); Letsinger, R. L. et al.,Nucleoside & Nucleotide 13: 1597 (1994); Chapters 2 and 3, ASC SymposiumSeries 580, “Carbohydrate Modifications in Antisense Research”, Ed. Y.S. Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic & MedicinalChem. Lett. 4: 395 (1994); Jeffs et al., J. Biomolecular NMR 34: 17(1994); Tetrahedron Lett. 37: 743 (1996)) and non-ribose backbones,including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, andChapters 6 and 7, ASC Symposium Series 580, “Carbohydrate Modificationsin Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acidscontaining one or more carbocyclic sugars are also included within thedefinition of nucleic acids (see Jenkins et al., Chem. Soc. Rev. 169-176(1995)); all citations are hereby expressly incorporated by reference.

In a preferred embodiment, the nucleic acids are peptide nucleic acids(PNA), which includes peptide nucleic acid analogs. These backbones aresubstantially non-ionic under neutral conditions, in contrast to thehighly charged phosphodiester backbone of naturally occurring nucleicacids. This results in several advantages. The PNA backbone exhibitsimproved hybridization kinetics. PNAs have larger changes in the meltingtemperature (T.sub.m) for mismatched versus perfectly matched basepairs.DNA and RNA typically exhibit a 2-4.degree. C. drop in T.sub.m for aninternal mismatch while with the non-ionic PNA backbone, the drop iscloser to 7-9.degree. C. Similarly, due to their non-ionic nature,hybridization of the bases attached to these backbones is relativelyinsensitive to salt concentration. Particularly preferred are PNAderivatives extendible by polymerase. These primers comprise PNAoligomers with an attached nucleotide that is recognized and extended bya polymerase (see Lutz, M. J. et al., Nucleosides Nucleotides 18:393-401 (1999) and Misra, H. S. Biochemistry 37: 1917-1925 (1998);publications hereby incorporated by reference). PNAs with a nucleotideor dinucleotide 3′ terminus are recognized and extended by severalpolymerases, and the presence of the PNA segment will render thePNA-modified strand resistant to nuclease, particularly a 5′-3′exonuclease.

Although primers are generally single stranded, the nucleic acids asdescribed herein may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, RNA, or hybrid, wherethe nucleic acid contains any combination of deoxyribo- andribonucleotides, and any combination of bases, including uracil,adenine, thymine, cytosine, guanine, xanthine hypoxanthine, isocytosine,isoguanine, inosine, etc., although generally occurring bases arepreferred. As used herein, the term “nucleoside” includes nucleotides aswell as nucleoside and nucleotide analogs, and modified nucleosides suchas amino modified nucleosides. In addition, “nucleoside” includesnon-naturally occurring analog structures. Thus, for example, theindividual units of a peptide nucleic acid (PNA), each containing abase, are referred herein as a nucleotide.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention. In addition, mixtures ofnaturally occurring nucleic acids and analogs or mixtures of differentnucleic acid analogs may be made.

The size of the primer nucleic acid may vary, as will be appreciated bythose in the art, in general varying from 5 to 500 nucleotides inlength, with primers of between 10 and 100 being preferred, between 12and 75 being particularly preferred, and from 15 to 50 being especiallypreferred, depending on the use, required specificity, and theamplification technique.

Generally, the compositions of the present invention provide for a firstprimer which hybridizes to a first single strand of the target nucleicacid and a second primer which hybridizes to a second single strand ofthe target nucleic acid, where the first and second strands aresubstantially complementary. The primers are capable of primer extensionby polymerase when hybridized to their respective strands. That is, theprimers hybridized to the target nucleic acid have their 3′ terminalnucleotide residues complementary to the nucleotide residue on thetarget nucleic acid such that the primers are capable of primerextension.

In one preferred embodiment, at least one of the primers furthercomprises at least one altered or mutated nucleotide residue, whichproduces a mismatch between the altered residue and the 3′ terminalnucleotide of the other primer when the primers hybridize to each other.By “altered”, “changed”, or “mutated” nucleotide residue herein is meantany change in the nucleotide residue of the primer, such astransversions and transitions, to generate the required mismatch. Thepresence of a mismatch at the 3′ terminal nucleotide blocks extension bypolymerase, thus limiting non-target nucleic acid dependent extensionreactions.

Accordingly, in one preferred embodiment, at least one nucleotide of thefirst primer is altered to produce a mismatch between the alteredresidue and the 3′ terminal nucleotide residue of the second primer whenthe first and second primers hybridize to each other.

For any primer pair, the ability of the primers to hybridize to eachother may be examined by aligning the sequence of the first primer tothe second primer. The stability of the hybrids, especially the thermalmelting temperature (T.sub.m), may be determined by the methodsdescribed below and by methods well known in the art. These include, butare not limited to, nearest-neighbor thermodynamic calculations (seeBreslauer, T. et al., Proc. Natl. Acad. Sci. USA 83: 8893-8897 (1986);Wetmur, J. G. Crit. Rev. Biochem. Mol. Biol. 26: 227-259 (1991);Rychlik, W. et al., J. NIH Res. 6: 78 (1994)); Wallace Rule estimations(Suggs, S. V. et al, “Use of Synthetic oligodeoxyribonucleotides for theisolation of specific cloned DNA sequences,” Developmental biology usingpurified genes, D. B. Brown, ed., pp 683-693, Academic Press, New York,1981), and T.sub.m estimations based on Bolton and McCarthy (seeBaldino, F. J. et al., Methods Enzymol. 168: 761-777 (1989); Sambrook,J. et al., Molecular Cloning: A Laboratory Manual, Chapter 10, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001). Allreferences are hereby expressly incorporated by reference. The effect ofvarious parameters including, among others, ionic strength, probelength, G/C content, and mismatches are taken into consideration whenassessing hybrid stability. Consideration of these factors are wellknown to those skilled in the art (Sambrook, J., supra).

Whether the hybrids formed between the primers are capable ofnon-template dependent extensions by polymerase is assessed by thepresence of complementarity at the 3′ terminal regions of the hybrids.Those hybrids capable of being extended by polymerase have at least the3′ terminal nucleotide of at least one of the primers complementary tothe nucleotide sequence of the other primer (Watson, R. Amplifications5-6 (1989)). Thus, hybrids comprising complementarity of at least the 3′terminal nucleotide of at least one of the primers is selected foralteration or mutation of the primer sequences. Since primer extensionby polymerase is less efficient when there is a mismatch near the 3′terminal nucleotide, alterations are preferred for primer hybrids withat least 2 or more nucleotides, and most preferably for hybrids with 3or more complementary nucleotides at the 3′ terminal region. Identifyinghybrids with complementarity at 3′ terminal regions may also be doneusing various algorithms, for example Amplify 1.2 software (Universityof Wisconsin, Department of Genetics, Madison, Wis.).

Alternatively, the presence of non-target nucleic acid dependent primerextension products are readily assessed by conducting amplificationreactions in the absence of target nucleic acid and examining theproducts for primer-dimer species, for example by electrophoresis. By“primer-dimer” as used herein is meant non-target nucleic acid dependentextensions by polymerase arising from primers hybridized to otherprimers. The presence of primer-dimer products may also be assessed byconducting the amplification reaction in the absence or presence oftarget nucleic acid and analyzing the melting curves of theamplification products (see Ririe, K. M. Anal. Biochem. 245: 154-160(1997); incorporated by reference).

In a preferred embodiment, the altered or mutated nucleotide residue issufficiently distant from the 3′ terminus of the primer to permitefficient extension by polymerase when primers hybridize to theirrespective strands of the target nucleic acid. Since it is well knownthat mismatches at or near the 3′ terminal nucleotide can interfere withprimer extension, the altered nucleotide residue is at least 1 or moreresidues from the 3′ terminal nucleotide of the altered primer. Morepreferably, the altered residue is at least 2 or more residues, while inthe most preferred embodiment, the altered residue is at least 3 or moreresidues from the 3′ terminal nucleotide.

In another preferred embodiment of the present invention, both primersused in, the amplification reaction comprise at least one alterednucleotide residue such that hybridization of primers to each othergenerates mismatches at the 3′ terminal residues of both primers toprevent extension by polymerase. This situation arises when each of theprimers have 3′ terminal regions complementary to the other primer.Thus, in a preferred embodiment, in addition to at least one alterednucleotide residue on the first primer, at least one nucleotide residueon the second primer is altered to produce a mismatch between thealtered residue on the second primer and the 3 terminal nucleotide ofthe first primer when the first and second primers hybridize to eachother. As discussed above, the altered residue is preferably at least 1nucleotide residue, more preferably at least 2 nucleotide residues, andmost preferably at least 3 nucleotide residues from the 3′ terminalnucleotide of the second primer. Since the 3′ terminal nucleotideresidues of the first and second primers are not altered, they remaincapable of being extended by polymerase when the primers are hybridizedto their respective strands of the target nucleic acid. Consequently,target nucleic acids are readily amplified while non-target nucleic acidproducts are not amplified.

Although the embodiments described above relate to two different primerscapable of hybridizing to each other, there are circumstances where aprimer will be complementary to itself, resulting in primer-dimerproducts of a single primer. Thus, the present invention is not limitedto different primers but also applicable to self-complementary primersthat give rise to primer-dimer products in amplification reactions.

In view of the general design of primers for limiting non-target nucleicacid dependent amplifications, the present invention further providesfor compositions useful for amplifying a plurality of repetitive unitsin a repetitive region of a target nucleic acid. By “repetitive unit”,“repeat unit”, “repetitive element” or grammatical equivalents as usedherein is meant a minimal nucleotide sequence which is reiterated orrepeated in the repetitive region. The repetitive unit for amplificationmay comprise any repetitive sequence, with repetitive units of 1 or morenucleotides being preferred, more preferably repetitive units between 3and 100 nucleotides, and most preferably repetitive units between 4 and30 nucleotides. In general, these repetitive units are arranged intandem fashion, although there may be non-repetitive nucleotides presentbetween the repetitive units. By “plurality” of repetitive elementsherein is meant at least two or more repetitive units in the repetitiveregion. The repetitive regions may comprise those existing in nature,such as repetitive regions comprising centromeres and telomeres, or theymay comprise repetitive regions introduced into cells and organisms, forexample by transfection, recombination, or site specific integration(e.g., retroviral delivery). The number of repetitive units amplifiedfor each set of primers will depend on the length of the primer and thenucleotide length of the repetitive unit. As will be appreciated bythose skilled in the art, primer sequences and primer lengths may bechosen based on stability and specificity of the primer for therepetitive units.

In a preferred embodiment, the compositions for amplifying repetitiveunits of a repetitive region comprises a first primer which hybridizesto at least one repetitive unit on a first single strand of the targetnucleic acid and a second primer which hybridizes to at least onerepetitive unit on a second single strand of the target nucleic acid,where the first and second strands are substantially complementary. Theprimers are capable of primer extension when hybridized to theirrespective strands of the target nucleic acid. That is, the primershybridized to the target nucleic acid have their 3′ terminal nucleotideresidues complementary to the nucleotide residue on the target nucleicacid such that the primers are capable of primer extension. In oneaspect, at least one nucleotide residue of at least one of the primersis altered to produce mismatches with a nucleotide residue of at leastone repetitive unit to which the primer hybridizes, wherein the alterednucleotide residue also produces a mismatch with the 3′ terminalnucleotide residue of the other primer when the primers hybridize toeach other, thus limiting primer extension of primer-primer hybrids.

Accordingly, in a preferred embodiment, at least one nucleotide residueof the first primer is altered to produce a mismatch between the alteredresidue and a nucleotide residue of at least one repetitive unit of thefirst strand to which the primer hybridizes, wherein the alterednucleotide residue also produces a mismatch with the 3′ terminalnucleotide residue of the second primer when the first and secondprimers hybridize to each other.

The altered nucleotide residue is preferably at least 1 nucleotideresidue, more preferably at least 2 nucleotide residues, and mostpreferably 3 nucleotide residues from the 3′ terminal nucleotide toallow efficient extension by polymerase when the altered primerhybridizes to target nucleic acids.

In another aspect, both primers used for amplifying repetitive unitscomprise at least one altered nucleotide residue such that hybridizationof primers to each other generates mismatches between the alteredresidues and the 3′ terminal residues of both primers. Thus, in apreferred embodiment, in addition to the altered nucleotide residue onthe first primer described above, at least one nucleotide residue of thesecond primer is altered to produce a mismatch between the alteredresidue and a nucleotide residue of at least one repetitive unit of thesecond strand to which the second primer hybridizes, wherein the alteredresidue on the second primer also produces a mismatch with the 3′terminal nucleotide of the first primer when the primers hybridize toeach other.

In yet another preferred embodiment for amplifying repetitive units of arepetitive region, the present invention comprises a first primer whichhybridizes to more than one repetitive unit on a first single strand ofa target nucleic acid and a second primer which hybridizes to more thanone repetitive unit on a second single strand of the target nucleicacid, where the first and second strands are substantiallycomplementary. The primers are capable of primer extension whenhybridized to their respective strands of the target nucleic acid, asdescribed above. In one aspect, nucleotide residues of at least one ofthe primers are altered to produce mismatches between the alteredresidues and the nucleotide residues at the identical nucleotideposition of each repetitive unit of the single strand of the targetnucleic acid to which the primer hybridizes. These altered nucleotideresidues also produce mismatches with the 3′ terminal nucleotide residueof the other primer when the primers hybridize to each other, thusfurther limiting primer-extension of primer-primer hybrids.

Accordingly, in one preferred embodiment, nucleotide residues of thefirst primer is altered to produce mismatches between the alteredresidues and nucleotide residues at the identical nucleotide position ofeach repetitive unit of the first strand of the target nucleic acid towhich the primer hybridizes. These altered nucleotides also producemismatches with the 3′ terminal nucleotide residue of the second primerwhen first second primer hybridize to each other.

The altered nucleotide residues are preferably at least 1 nucleotideresidue, more preferably at least 2 nucleotide residues, and mostpreferably 3 nucleotide residues from the 3′ terminal nucleotide toallow efficient extension by polymerase when the altered primerhybridizes to target nucleic acids.

In another aspect, both primers comprise altered nucleotide residuessuch that hybridization of primers to each other result in mismatches ofthe 3′ terminal nucleotide of both primers. Thus, in a preferredembodiment, in addition to the altered nucleotide residues on the firstprimer, nucleotide residues on the second primer are altered to producemismatches between the altered residues of the second primer andnucleotide residues at the identical nucleotide position of eachrepetitive unit of the second strand to which the primer hybridizes.These altered nucleotides of the second primer also produce mismatcheswith the 3′ terminal nucleotide residue of the first primer when theprimers hybridize to each other.

Since primers hybridized to target nucleic acids must be capable ofprimer extension, alterations of the first and second primers must be onnon-complementary nucleotides of the repetitive unit. Thus, in oneaspect, when both the first and second primers comprise alteredresidues, the alterations are at adjacent nucleotide positions of therepetitive unit. In another aspect, the alterations are situated onnon-adjacent nucleotide positions of the repetitive unit. In general,mismatches at adjacent nucleotide positions provide for the most numberof base paired or complementary residues between the altered nucleotideand the 3′ terminal nucleotide, which may be important for efficientlyamplifying short repetitive sequences (i.e., 3-6 basepairs).

In another preferred embodiment, the first and second primers furthercomprise a 5′ terminal region that does not hybridize (i.e., basepair)with the target nucleic acid. The unpaired region comprises one or morenucleotides, with a preferred range of 3 to 60 nucleotides, and a mostpreferred range of 4 to 30 nucleotides. When the primers are directedtowards amplification of repetitive units of a repetitive region, the 5′unpaired region blocks the 3′ ends of the replicated primer extensionproducts from initiating nucleic acid synthesis from the internalrepetitive units of the amplification products during subsequentamplification cycles.

Although the 5′ terminal unpaired region may be of any sequence whichdoes not hybridize to the target nucleic acid, in a preferred embodimentthe unpaired region comprises restriction sites, unique sequences forpurposes of sequencing or primer extension reactions (i.e.amplification), or tag sequences for detecting and measuring theamplified product.

In a preferred embodiment, the primers of the present invention aredesigned to have similar T_(m)s. As used herein, primers with similarT_(m)s have a T_(m), difference of about 10° C. or less, preferably 5°C. or less, and more preferably 2° C. or less. Use of primer sets (e.g.,primer pairs) with similar or identical T_(m)s allow use of anannealing/extension temperature optimal for both primers and providessimilar amplification efficiency at a particular amplificationcondition. Advantages are the ability to use similar concentrations ofprimers, particularly at lower concentrations, which limits generationof unwanted amplification products. By comparison, when T_(m)s of theprimers are dissimilar, one primer is used at a higher concentration tocompensate for differences in amplification efficiency. This higherprimer concentration results in undesirable amplification products at alower number of PCR cycles.

In one aspect, primers with similar T_(m)s are made by altering thelength of primers or by selecting primers having similarguanosine-cytosine (GC) content. T_(m)s are assessed by the methodsdescribed above. As used herein, a “similar GC content” is meant aprimer set which has a GC content difference of about 10% or less, morepreferably a difference of about 5% or less, and most preferably adifference of about 2% or less, such that the primers display similarT_(m)s, as defined above. In the primer design process, T_(m) and/or GCcontent is initially assessed for the region that hybridizes to thetarget nucleic acid. For primers with a non-hybridizing 5′ terminalregion described above, additional analysis of the T_(m) and GC contentis conducted for the entire primer sequence. Generally, primers aredesigned to have higher similarities of GC content at the 3′ terminalregion since it is this region that is extended by polymerase.

The primers of the present invention may be used to amplify varioustarget nucleic acids. A single primer set, for example a primer pair,may be used to amplify a single target nucleic acid, or multiple primersets may be used to amplify a plurality of target nucleic acids.Amplifications may be conducted separately for each unique primer set,or in a single reaction vessel using combinations of primer sets,generally known in the art as multiplexing. When multiple primer setsare used in a single reaction, primers are designed to limit formationof undesirable products and limit interference between primers of eachprimer set.

As the present invention relates to amplifying target nucleic acids withthe primers described above, the present invention further provides formethods of amplifying target nucleic acid sequences. In a preferredembodiment, the method comprises contacting a target nucleic acidcomprising substantially complementary first and second strands with thefirst and second primers described above, and amplifying the targetnucleic acid by polymerase chain reaction.

By “target nucleic acid” or “target sequence” or grammatical equivalentsherein is meant a nucleic acid sequence on a double or single strandednucleic acid. The target sequence may be a portion of a gene, aregulatory sequence, genomic DNA, cDNA, RNA, including mRNA and rRNA, orother nucleic acids. It may be any length, with the understanding thatlonger sequences are more specific. In some embodiments, it may bedesirable to fragment or cleave the sample nucleic acid into fragmentsof 100-10,000 base pairs, with fragments of roughly 500 basepairs beingpreferred in some embodiments. Fragmentation or cleavage may be done inany number of ways well known to those skilled in the art, includingmechanical, chemical, and enzymatic methods. Thus, the nucleic acids maybe subjected to sonication, French press, shearing, or treated withnucleases (e.g., DNase, restriction enzymes, RNase, etc.), or chemicalcleavage agents (e.g., acid/piperidein, hydrazine/piperidine, iron-EDTAcomplexes, 1,10-phenanthroline-copper complexes, etc.).

As will be appreciated by those skilled in the art, the target sequencemay take many forms. For example it may be contained within a largernucleic acid sequence, i.e., all or part of a gene or mRNA, arestriction fragment of a plasmid or genomic DNA, among others. Thesample comprising the target sequence may be obtained from any tissue ofany organism, including blood, brain, bone marrow, lymnph, liver,spleen, breast, epithelia (e.g., skin, mouth, etc.), or other tissues,including those obtained from biopsy. The samples may also comprisebodily excretions or fluids, such as saliva, urine, feces, cerebrospinalfluid, semen, milk etc. Other sources of target nucleic acids includebacteria, yeast, plant, virus, or other nucleic acid containingorganisms, pathogenic or nonpathogenic. The nucleic acid can also be anynucleic acid generated artificially by chemical or enzymatic processes,such as PCR reactions.

The target sequence may be prepared using well known techniques. Forinstance, the sample may be treated using detergents, sonication,electroporation, denaturants, etc., to disrupt the cells, bacteria, orviruses. The target nucleic acids may be purified as needed. Componentsof the reaction may be added simultaneously, or sequentially, in anyorder as outlined below. In addition, a variety of agents may be addedto the reaction to facilitate optimal hybridization, amplification, anddetection. These include salts, buffers, neutral proteins, detergentsetc. Other agents may be added to improve efficiency of the reaction,such as protease inhibitors, nuclease inhibitors, ant-microbial agents,etc., depending on the sample preparation methods and purity of thetarget nucleic acid. When the target nucleic acid is RNA, these nucleicacids may be converted to DNA, for example by treatment with reversetranscriptase (e.g., MoMuLV reverse transcriptase, Tth reversetranscriptase, etc.), as is well known in the art.

When the target nucleic acids are double stranded nucleic acids, theyare denatured to generate a first single strand and a second singlestrand so as to allow hybridization of the primers. Any number ofdenaturation steps may be used such as temperature to about 95° C.although alkaline pH, denaturants (e.g., formamide), and othertechniques may be applied as appropriate to the nature of the doublestranded nucleic acid.

The primers are contacted with the target nucleic acid so that a firstprimer is capable of hybridizing to a first strand and a second primeris capable of hybridizing to a second strand of the target nucleic acid.A variety of hybridization conditions may be used to form the hybrids,including high, moderate, and low stringency conditions (see forexample, Sambrook, J., Molecular Cloning: A Laboratory Manual, 3rd ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001;Ausubel, F. M. et al., Current Protocols in Molecular Biology, JohnWiley & Sons, updates to 2001; all of which are hereby incorporated byreference). Stringency conditions are sequence-dependent and will bedifferent in different circumstances, including length of primer, numberof mismatches, G/C content, and ionic strength. A guide to hybridizationof nucleic acids is provided in Tijssen, P., “Overview of Principles ofHybridization and the Strategy of Nucleic Acid Assays,” in LaboratoryTechniques in Biochemistry and Molecular Biology: Hybridization withNucleic Acid Probes, Vol. 24, Elsevier, Amsterdam, 1993). Generally,stringent conditions are selected to be about 5-10.degree. C. lower thanthe thermal melting point (T_(m)) for a specific hybrid at a definedsolution condition (e.g., ionic strength, pH, concentration of nucleicacid). The T_(m) is defined as the temperature under a defined solutioncondition at which 50% of the primer sequences complementary to thetarget nucleic acid are hybridized, or single stranded, at equilibrium.Generally, the solution condition is the solution conditions used foramplifying the target nucleic acids. Since the degree of stringency isgenerally the difference in the temperature of hybridization and theT_(m), the degree of stringency may be maintained despite changes insolution condition of hybridization as long as the difference intemperature from T_(m) is maintained. The hybridization conditions mayalso vary with the type of nucleic acid backbone, for exampleribonucleic acid or peptide nucleic acid (PNA) backbone.

In hybridizing the primers to the target nucleic acids and in theamplification reactions, the assays are generally done under stringencyconditions that allow formation of the hybrids in the presence of targetnucleic acid. Those skilled in the art can alter the parameters oftemperature, salt concentration, pH, organic solvent, chaotropic agents,or other variables to control the stringency of hybridization and alsominimize hybridization of primers to non-specific targets (e.g., by useof “hot start” PCR or “touchdown” PCR).

Following contacting the primers to the target nucleic acids, thereaction is treated with an amplification enzyme, generally apolymerase. A variety of suitable polymerases are well known in the art,including, among others, Taq polymerase, KlenTaq, Tfl polymerase,DynaZyme, etc. Generally, although all polymerases are applicable to thepresent invention, preferred polymerases are thermostable polymeraseslacking 3′ to 5′ exonuclease activity since use of polymerases withstrong 3′ to 5′ exonuclease activity tends to remove the mismatched 3′terminal nucleotides. Also useful are polymerases engineered to havereduced or non-functional 3′ to 5′ exonuclease activities (e.g.,Pfu(exo-), Vent(exo-), Pyra(exo-), etc.). Also applicable are mixturesof polymerases used to optimally extend hybridized primers. In anotheraspect, polymerase enzymes useful for the present invention areformulated to become active only at temperatures suitable foramplification. Presence of polymerase inhibiting antibodies, whichbecome inactivated at amplification temperatures, or sequestering theenzymes in a form rendering it unavailable until amplificationtemperatures are reached, are all suitable. These polymeraseformulations allow mixing all components in a single reaction vesselwhile preventing priming of non-target nucleic acid sequences.

In another aspect, those skilled in the art will appreciate that variousagents may be added to the reaction to increase processivity of thepolymerase, stabilize the polymerase from inactivation, decreasenon-specific hybridization of the primers, or increase efficiency ofreplication. Such additives include, but are not limited to, dimethylsulfoxide, formamide, acetamide, glycerol, polyethylene glycol, orproteinacious agents such as E. coli. single stranded DNA bindingprotein, T4 gene 32 protein, bovine serum albumin, gelatin, etc. Inanother aspect, the person skilled in the art can use various nucleotideanalogs for amplification of particular types of sequences, for exampleGC rich or repeating sequences. These analogs include, among others,c.sup.7-dGTP, hydroxymethyl-dUTP, dITP, 7-deaza-dGTP, etc.

Amplification reactions are carried out according to procedures wellknown in the art. Procedures for polymerase chain reaction are widelyused and described (see for example, U.S. Pat. Nos. 4,683,195 and4,683,202; hereby incorporated by reference). In brief, a doublestranded target nucleic acid is denatured, generally by incubating at atemperature sufficient to denature the strands, and then incubated inthe presence of excess primers, which hybridizes (i.e., anneals) to thesingle-stranded target nucleic acids. A DNA polymerase extends thehybridized primer, generating a new copy of the target nucleic acid. Theresulting duplex is denatured and the hybridization and extension stepsare repeated. By reiterating the steps of denaturation, annealing, andextension in the presence of a second primer for the complementarytarget strand, the target nucleic acid encompassed by the two primers isexponentially amplified. The time and temperature of the primerextension step will depend on the polymerase, length of target nucleicacid being amplified, and primer sequence employed for theamplification. The number of reiterative steps required to sufficientlyamplify the target nucleic acid will depend on the efficiency ofamplification for each cycle and the starting copy number of the targetnucleic acid. As is well known in the art, these parameters can beadjusted by the skilled artisan to effectuate a desired level ofamplification. Those skilled in the art will understand that the presentinvention is not limited by variations in times, temperatures, bufferconditions, and amplification cycles applied in the amplificationprocess.

The products of the amplification are detected and analyzed by methodswell known in the art. Amplified products may be analyzed followingseparation and/or purification of the products, or by direct measurementof product formed in the amplification reaction. Separation andpurification methods include, among others, electrophoresis, includingcapillary electrophoresis (e.g., in agarose or acrylamide gels);chromatography (e.g., affinity, molecular sieve, reverse phase, etc.);and hybridization. The purified products may be subjected to furtheramplifications as is well known in the art. For detection, the productmay be identified indirectly with fluorescent compounds, for examplewith ethidium bromide or SYBR™Green, or by hybridization with labelednucleic acid probes. Alternatively, labeled primers or labelednucleotides are used in the amplification reaction to label theamplification product. The label comprises any detectable moiety,including fluorescent labels, radioactive labels, electronic labels, andindirect labels such as biotin or digoxigenin. When indirect labels areused, a secondary binding agent that binds the indirect label is used todetect the presence of the amplification product. These secondarybinding agents may comprise antibodies, haptens, or other bindingpartners (e.g., avidin) that bind to the indirect labels. Secondarybinding agents are preferably labeled with fluorescent moieties,radioactive moieties, enzymes, etc.

In another preferred embodiment, the amplification product may bedetected and quantitated during the amplification reaction by real timequantitative PCR, variations of which are well known in the art. Forinstance, the TaqMan system uses a probe primer which hybridizes tosequences in an internal sequence within the nucleic acid segmentencompassed by the primers used to amplify the target nucleic acid(Heid, C. A. et al., Genome Res. 6: 986-994 (1996); Holland, P. M. etal., Proc. Natl. Acad. Sci. USA 88: 7276-7280 (1991); incorporated byreference). This probe is labeled with two different flourescent dyes(i.e., dual-labeled fluorogenic oligonucleotide probe), the 5′ terminusreporter dye (TAMRA) and the 3′ terminus fluorescence quenching dye(FAM). Cleavage of the probe by the 5′ to 3′ exonuclease activity of DNApolymerase during the extension phase of PCR releases the fluorogenicmolecule from proximity of the quencher, thus resulting in increasedfluorescence intensity.

In another aspect, real time quantitative PCR may be based onfluorescence resonance energy transfer (FRET) between hybridizationprobes (Wittwer, C. T. Biotechniques 22: 130-138 (1997); incorporated byreference). In this method, two oligonucleotide probes hybridize toadjacent regions of the target nucleic acid sequence. The upstream probeis labeled at the 3′ terminus with an excitor dye (e.g., FITC) while theadjacently hybridizing downstream probe is labeled at the 5′ terminuswith a reporter dye. Hybridization of the two probes to the amplifiedtarget nucleic acid sequences positions the two dyes in close spatialproximity sufficient for FRET to occur. This allows monitoring thequantity of amplified product during the polymerase chain reaction. Asimilar approach is used in the molecular beacon probes (Tyagi, S, Nat.Biotechnol. 16: 49-53 (1998); incorporated by reference). Molecularbeacons are oligonucleotide probes comprising a quencher dye and areporter dye at the opposite ends of a PCR product specificoligonucleotide. The dyes may also function based on FRET, and thereforemay also be comprised of an excitation dye and a reporter dye. Shortcomplementary segments at the 5′ and 3′ terminal regions allow forformation of a stem-loop structure, which positions the dyes at theterminal ends of the oligonucleotide into close proximity, thusresulting in fluorescence quenching or FRET. When the oligonucleotidehybridizes to a PCR product through complementary sequences in theinternal region of the molecular beacon probe, fluorescence of theoligonucleotide probe is affected, thus allowing monitoring of productsynthesis.

Real time quantitative PCR may also use fluorescent dyes thatpreferentially bind to double stranded nucleic acid amplificationproducts during the PCR reaction, thereby providing continuousmonitoring of product synthesis (see Higuchi, R. et al., Biotechnology11: 1026-1030 (1993); Morrison, T. B. et al., Biotechniques 24: 954-962(1998)). Suitable fluorescent dyes include, among others, ethidiumbromide, YO PRO-1™ (Ishiguro, T. Anal Biochem. 229: 207-213 (1995)), andSYBR™. Green dyes (Molecular Probes, Eugene, Oreg., USA). Whenamplifying target nucleic acids comprising repetitive regions, FRET ormolecular beacon based probes are not preferred if FRET or molecularbeacon probes are directed to repetitive units since they will hybridizeto repetitive sequences on the primers, thereby failing to distinguishbetween primers and amplified product.

In a further preferred embodiment; real time quantitative PCR isaccomplished with primers containing a single fluorophore attached nearthe 3′ terminal nucleotide (Nazarenko, I. et al., Nucleic Acids Res. 30:e37 (2002); Nazarenko, I. et al., Nucleic Acids Res. 30: 2089-2195(2002); LUX™ Fluorogenic Primers, Invitrogen, Palo Alto, Calif.; herebyincorporated by reference). The 5′ end of these primers have a 5 to 7nucleotide extension capable of hybridizing to the 3′ terminal region togenerate a blunt-ended hairpin (i.e., stem-loop) structure, whoseformation results in fluorescence quenching of the fluorophore. When theprimer forms a duplex, for example by primer extension on a template,the quenching is reduced or eliminated, thus providing a measure of PCRproduct in the sample. Because only a single fluorphore is used,different fluorophores may be used and detected in a single reaction.Consequently, these primers are useful for amplification and detectionof a plurality of different target nucleic acids in a single reactionvessel by use of different primer sets with distinguishablefluorophores. As discussed herein, various target nucleic acids includecombinations of single copy genes and repetitive sequences.

Instrumentation suitable for real time monitoring of PCR reactions areavailable for use in quantitative PCR methods (ABI Prism 7700, AppliedBiosystems Division, Perkin Elmer, Fosters City, Calif., USA;LightCycler™, Roche Molecular Biochemicals, Indianapolis, Ind., USA).

When real time quantitative PCR is used to detect and measure theamplification products, various algorithms are used to calculate thenumber of target nucleic acids in the samples (see ABI Prism 7700Software Version 1.7; Lightcycler™ Software Version 3; incorporated byreference). Quantitation may involve use of standard samples with knowncopy number of the target nucleic acid and generation of standard curvesfrom the logarithms of the standards and the cycle of threshold (C_(t)).In general, C_(t) is the PCR cycle or fractional PCR cycle where thefluorescence generated by the amplification product is severaldeviations above the baseline fluorescence (Higuchi, R. et al., supra).Real time quantitative PCR provides a linearity of about 7 to 8 ordersof magnitude, which allows measurement of copy number of target nucleicacids over a wide dynamic range. The absolute number of target nucleicacid copies can be derived from comparing the C_(t) values of thestandard curve and the samples.

The copy number of target nucleic acids may also be determined bycomparative quantitative real time PCR. Use of nucleic acids of knowncopy number or consistent copy number allows quantitating the copynumber of target nucleic acids in a sample. The standard may be a singlecopy gene, a nucleic acid of known copy number, or when quantitating RNAcopy number, a constitutively expressed housekeeping gene (see Johnson,M. R. Anal. Biochem. 278: 175-184 (2000); Boulay, J.-L., et al.,Biotechniques 27: 228-232 (1999)).

The compositions and methods described above find use in any process foramplifying target nucleic acids by polymerase chain reaction. Thus, thepresent invention is useful in detecting and monitoring infectiousdiseases, for example in testing for presence of pathogenic bacteria andviruses (e.g., viral load). For instance, target viral nucleic acidsinclude, without limitation, HIV, HCV cytomegalovirus, hepatitis, etc.The present invention is also applicable for monitoring medicaltherapies. For example this may involve monitoring the progress ofbacterial infections following antibiotic administration.

The present invention finds applications in characterizing thefunctional state of cells, especially for cell changes associated withdisease states. For example, amplification of particular geneticsegments during cancer progression in ovarian or breast cancer iscorrelated not only with the stage of the cancer but also survival rates(see Kalioniemi, A et al., Proc. Natl. Acad. Sci. USA 91: 2156-2160(1994)). Thus, quantitation of gene amplification has diagnostic andprognostic value for a variety of tumors (Kawate, S. et al., Oncology57: 157-163 (1999); Biechi, I. et al., Int. J. Cancer 78: 661-666(1998)). Conversely, loss of genetic elements (i.e., loss ofheterozygosity or microsatellite instability) is also associated withparticular disease states, thus providing a useful focal point fordisease diagnostics. For example, deletions of chromosomal region 18q21is frequently observed in colorectal cancers while the tumor suppressorp53 gene located on chromosome 17p13.1 is frequently deleted in avariety of cancer types (Largey, J. S. et al., Cancer 17: 1933-1937(1993)).

Of particular importance in cell function and disease are repetitivesequences, especially tandemly repetitive sequences found in genomes ofmany organisms. In eukaryotes, these sequences are generally classifiedinto three major categories: satellite, minisatellite, andmicrosatellite. Satellite DNAs have repeat lengths of about 1 to severalthousand bases and can constitute repetitive regions of up to 100million basepair clusters, for example in heterochromatic regions ofeukaryotic chromosomes. These sequences are mainly associated withcentromeres and telomeres. Minisatellite sequences are moderatelyrepetitive, tandemly repeated arrays of about 9-100 basepair repeats,generally having mean array lengths of about 0.5 to 30 kb. These aregenerally found in euchromatic regions and are highly variable in size.Microsatellites are moderately repetitive arrays of short (i.e., about2-6 basepairs) repeats. Copy numbers of these repeats vary within apopulation, typically having mean array sizes of about 10 to 100 bases.In many cases, changes in the extent of repeats is often correlated withcertain diseases. For example, increase in the copy number of tripletrepeats CGG, CTG, and GAA is associated with Huntington's disease,Fragile X syndrome, and myotonic dystrophy. The extent of the expansionof these triplet repeats is often associated with the severity or onsetof the disease state such that progeny inheriting the expanded repeatshave more severe disorders or, where the disorder is not congenital, anearlier age of onset. Disorders related to expansion of tandem repeatsequences is not limited to trinucleotide repeats and can involve largerrepeating units (see Lafreniere, R. G. et al., Nat. Genet. 15: 298-302(1997)).

Tandem repetitive sequences also have important biological functions.With the certain exceptions (e.g., budding yeast), the centromeres ofmost plants, animal and fungus have large arrays of tandem repetitivesequences. Although the physiological roles of these sequences areuncertain, it is believed that they function in the assembly of thekinetichore to ensure faithful and efficient chromosome segregation.Thus, determining the variability in the number of repeats can provideinformation about function and regulation of centromeres and diseasesrelated to centromere dysfunction.

Of more defined importance in cell function are tandemly repeatingsequences comprising the telomeres of linear eukaryotic chromosomes.Telomeric DNA or telomeric region is the chromosomal region located atthe ends of chromosomes, consisting of tandem arrays of repetitive unitsof short sequences ranging from about 5 to 26 basepairs. The telomericregions of different organisms differ in their repetitive unit or repeatsequence. These repeat sequences are known for a variety of organisms,including human and other mammals, Tetrahymena, yeast, Drosophila, andnematodes. In humans, the telomeric repetitive unit is 5′-TTAGGG-3′while the Tetrahymena repetitive unit is 5′-TTGGGG-3′.

The telomere repetitive unit not only varies between species as to therepeat sequence, but also as to number of repetitive units in anorganism. It is well established that the length and integrity oftelomeres is important for cell growth and proper segregation ofchromosomes. For example, development of many types of cancerscorrelates with activation of telomere maintenance while cellsenescence, a condition in which cells have lost the ability toreplicate, although the normal replicative signals are present,correlate with loss of telomere integrity. For example, shortening oftelomere induces proliferative senescence in cells while telomeraseinhibition can lead to induced cell apoptosis (Zhang, X. et al., GenesDev. 2388-2399 (1999)). Moreover, knockout of telomerase RNA in miceresults in animals with developmental defects, age related pathologies,and increased cancer susceptibility (Rudolph, K. L. et al., Cell 96:701-712 (1999); Herrera, E. et al., EMBO J. 18: 2950-2960 (1999)).

Thus, measuring the number of repetitive units of specific repetitivesequences find important applications, including, but not limited to,cancer diagnosis, diagnosis of aging related diseases, integrity ofcloned organisms, screening of inherited disorders, and drug screeningfor agents directed to enzymes (i.e., telomerase) and cellular pathwaysregulating length of repetitive sequences.

Thus, in a preferred embodiment, the present invention provides forrapid analysis of telomere lengths by direct amplification of the repeatsequences using primers incapable of generating primer-dimers butcapable of primer extension when hybridized to telomere repetitiveunits. Since the telomeres of various organisms have differingrepetitive unit sequences, amplifying telomeres of a specific organismwill employ primers specific to the repetitive unit of the organism.Human telomeric sequences are used herein to illustrate practice of thepresent invention for direct amplification and quantitation of tandemlyrepeated nucleic acid sequences, but is not limited to the specificembodiment described herein.

In determining the number of telomeric repetitive units, the selectedprimers are complementary to repetitive units of the repetitive region.The first primer has sequences complementary to telomeric repetitivesequences on a first single strand of the target nucleic acid, and asecond primer has sequences complementary to the telomeric repetitivesequences on a second single strand of the target nucleic acid, whereinthe first and second strands are substantially complementary. In onepreferred embodiment, nucleotide residues of the first primer arealtered to produce mismatches between the altered residues andnucleotide residues at the identical nucleotide position of eachtelomere repetitive unit of the first strand of the target nucleic acid.These altered residues also produce a mismatch with the 3′ terminalnucleotide of the second primer when the primers hybridize to eachother. In another preferred embodiment, the nucleotide residues of thesecond primer are also similarly altered such that hybridization primersto each other results in the first and second primers with mismatched 3′terminal nucleotides.

In the preferred embodiment, the altered nucleotide residues producingthe mismatches are on both the first and second primers to limitformation any primer-dimer products. In this arrangement, the mismatchesmay be on adjacent or on non-adjacent nucleotide positions of therepetitive unit. Mismatches on adjacent nucleotide positions of therepetitive unit maximizes the number of basepaired residues from the 3′terminal nucleotide to the altered residue of each primer. Anexemplification of primers for amplification of human telomererepetitive units are provided in FIG. 1.

Subsequent to contacting the first and second primers to the singlestranded form of the repetitive region, the primers are extendedpolymerase, and the repetitive units amplified by reiterative cycles ofdenaturation, annealing, and extension. In a preferred embodiment, the5′ terminal region comprises non-basepairing sequences to restrictpriming from internal repeats of amplified repetitive sequences.

The amplified products are quantitated as described above. In apreferred embodiment, real time quantitative PCR is used to determinethe copy number of the telomere repetitive units in the target nucleicacid sample. Standards for determining and comparing telomere repetitiveunit number include use of single copy genes (e.g., ribosomalphosphoprotein 364B) or a target nucleic acid of known copy number(e.g., a plasmid with known number of telomere repetitive units). By themethods described herein, the copy number of repetitive units of a largenumber of samples may be quantitated for purposes of determining thenumber of telomere repetitive units, and thus the average length oftelomeres.

Measuring the number of repetitive units of telomeres has a wide varietyof applications in medical diagnosis, disease prognosis, andtherapeutics. The present invention is useful for determining telomerelengths of various types of cancer cells since activation of telomeraseactivity is associated with immortalization of cells. Cells can beanalyzed over time to determine whether an increase, decrease, orstabilization of telomeres is associated with disease progression.Various cancer cell types amenable for testing include breast, liver,brain, bone, prostate, lymphocyte, melanoma, colon cancers, etc.

The present invention also finds use in diagnosis of diseases related toearly onset of aging. For example, individuals with Hutchinson Gilfordprogeria disease show premature aging and reduction in proliferativepotential in fibroblasts associated with loss of telomeric length(Alssopp, R. C. et al., Proc. Natl. Acad. Sci. USA 89: 10114-10118(1992)) while patients with dyskeratosis congenita display progressivebone-marrow failure, abnormal skin pigmentation, leukoplakia, and naildystrophy because of a deletion of telomerase RNA (see Vulliamy, T.Nature 413: 432-435 (2001)). Thus, amplification and quantitation of thenumber of telomeric repeats is useful for determining the association ofparticular diseases with changes in telomere length.

In another preferred embodiment, the present invention is useful inmonitoring effectiveness of therapeutics or in screening for drugcandidates affecting telomere length or telomerase activity. Forexample, the present invention finds use in monitoring the effectivenessof cancer therapy since the proliferative potential of cells may berelated to the maintenance of telomere integrity. The ability to monitortelomere characteristics can provide a window for examining theeffectiveness of particular therapies and pharmacological agents. Inanother aspect, the present invention finds use as a general method ofscreening for candidate drugs affecting biological pathways regulatingtelomere length, such as telomerase activity. Ability to rapidly amplifytelomere repeats provides a high thoroughput screening method foridentifying small molecules, candidate nucleic acids, and peptidesagents affecting telomere characteristics in the cell.

It is understood by the skilled artisan that the steps for constructingthe primers and the methods for amplifying target nucleic acid sequencescan be varied according to the options provided herein. The followingexamples serve to more fully describe the manner of using and the bestmode for the above described invention. It is also understood, however,that these embodiments in no way serve to limit the scope of the presentinvention and that those skilled in the art may modify according to theskill in the art. All references cited herein are incorporated byreference.

EXAMPLES Example 1 Direct Amplification of Human Telomeric RepetitiveSequences

Genomic DNA was extracted from blood samples by standard procedures. Thesamples used to compare quantitative PCR vs. Southern blot approaches totelomere measurement were donated by 21 unrelated individuals (11 womenand 10 men, age range 61-94 years) from Utah families that are part ofthe Centre pour les Etudes du Polymorphisme Humaine (CEPH) collectionused worldwide for building the human genetic linkage map (White, R. etal., Nature 313: 101-105 (1985)). Purified DNA samples were diluted in96-well microtiter source plates to approximately 1.75 ng/ul in 10 mMTris-HCl, 0.1 mM EDTA, pH 7.5 (final volume 300 ul per well), heated to95.degree. C. for 5 minutes in a thermal cycler, quick-chilled bytransfer to an ice-water bath for 5 minutes, centrifuged briefly at700.times.g, sealed with adhesive aluminum foil, and stored at 4.degree.C. until the time of assay.

Real time quantitative PCR on the extracted DNA samples are performed onseparate 96 well plates. Two master mixes of PCR reagents were prepared,one with the telomere (T) primer pair, the other with the single copygene (S) primer pair. Depending on the reaction conditions, 30 or 10 ulof T master mix was added to each sample well and standard curve well ofthe first plate, and 30 or 10 ul of S master mix was added to eachsample well and standard curve well of the second plate. For eachindividual in whom the T/S ratio was assayed, three identical 20 ulaliquots of the DNA sample (35 ng per aliquot) were added to plate 1,and another three aliquots were added to the same well positions inplate 2. For each standard curve, one standard DNA sample was dilutedserially in TE (10 mM Tris, 1 mM EDTA, pH 7.0) by .about.1.68-fold perdilution to produce five concentrations of DNA ranging from 0.63 ng/ulup to 5 ng/ul, which were then distributed in 20 ul aliquots to thestandard curve wells on each plate. The plates were then sealed with atransparent adhesive cover, centrifuged briefly at 700×g, and stored at4° C. in the dark until the PCR was performed (0-3 days later).

PCR amplification conditions depended on the primers used and thetemplate DNA being amplified. In one set of experiments, telomere repeatsequences were amplified with primer set tel 1(5′-GGTTTTTGAGGGTGAGGGTGAG-GGTGAGGGTGAGGGT-3′) (SEQ ID NO: 1) and tel 2(5′-TCCCGACTATCCCTATCCCTATCCC— TATCCCTATCCCTA-3′) (SEQ ID NO: 2).

Concentrations of reagents in the PCR mixes were 150 nM 6-ROX and0.2.times. SYBR™ Green I (Molecular Probes, Inc.); 15 mM Tris-HCl, pH8.0; 50 mM KCl; 2 mM MgCl₂; 0.2 mM of each dNTP; 5 mM D17; 1% DMSO; 1.25units of AmpliTaq Gold DNA polymerase (Applied Biosystems, Inc.); 270 nMof tel 1 primer; and 900 nM of tel 2 primer in a final volume of 50 ul.The thermal cycling profile began with a 95° C. incubation for 10 min.to activate the AmpliTaq Gold DNA polymerase followed by 18 cycles of95° C.×15 s and 54° C.×2 min.

Alternatively, the telomere specific primer sequences were optimized tohave similar T_(m)s, particularly by designing the primers to havesimilar or identical GC content. Each primer of primer set tel 1b:(5′-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3′) (SEQ ID NO: 8) and tel2b: (5′-GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3′) (SEQ ID NO: 9)carries a purposefully introduced single base substitution at the sixthbase from the 3′ end and at every sixth base thereafter in the 5′direction, for a total of five introduced base changes at every sixthposition of the repeat sequence. When the primers hybridize to thetarget telomeric DNA, five single base mismatches result but the hybridshave perfect complementarity to the target telomeric DNA sequences inthe last five bases at the 3′ end of each primer. Hybridization of theprimers to each other results in base-pairing at four out of sixpositions, and the 3′ terminal residue of each primer forms a mismatchwith the other primer. PCR conditions with these T_(m), optimizedprimers were 0.4.times. Sybr Green I, 15 mM Tris-HCl, pH 8.0; 50 mM KCl;1.5 mM MgCl₂, 1% DMSO, 2.5 mM DTT, 200 uM each dNTP, 0.75 Units ofAmpliTaq Gold DNA polymerase, 450 nM of tel 1 b primer, and 450 nM oftel 2b primer, in a final volume of 30 ul per reaction. The thermalcycling profile was 95° C.×10 min. followed by 18 cycles of 95° C. for15 sec. (denaturation) and 56° C. for 2 min. (anneal/extend). No ROX dyeis needed for this assay condition. In general, three telomere PCRreactions were done on each experimental DNA. Each 96 well telomere PCRplate contained a row with 2 NTC wells (no genomic DNA), and 10 standardcurve wells containing a serial dilution of a reference DNA to fiveconcentrations, with each concentration in duplicate, ranging from 0.25ng/microliter (final concentration) up to 2 ng/microliter (finalconcentration).

The 36B4 gene, which encodes acidic ribosomal phosphoprotein PO, wasused to normalize the telomere signal (Boulay et al., Biotechniques 27:228-232 (1999)). The primer set used was 36B4u(5′-CAGCAAGTGGGAAGGTGTAATCC-3′, SEQ ID NO: 6) and 36B4d,(5′-CCCATTCTATCATCAACGGGTACAA-3′, SEQ ID NO: 7). PCR conditionscontained 150 nM 6-ROX and 0.2×SYBR™Green I (Molecular Probes, Inc.); 15mM Tris-HCl, pH 8.0; 50 mM KCl; 2 mM MgCl₂; 200 uM of each dNTP; 5 mMDTT; 1% DMSO; 1.25 units of AmpliTaq Gold DNA polymerase (AppliedBiosystems, Inc.); 300 nM of 36B4u primer; and 500 nM. of 36B4d primerin a final volume of 50 ul per reaction. The thermal cycling profile was95° C. incubation for 10 min followed by 30 cycles of 95° C.×15 s, 58°C.×1 min. Alternatively, for optimal amplifications, the PCR reactionscontained 0.4.times. Sybr Green I, 15 mM Tris-HCl, pH 8.0; 50 mM KCl;3.5 mM MgCl₂, 1% DMSO, 2.5 mM DTT, 200 uM each dNTP, 0.75 Units ofAmpliTaq Gold DNA polymerase, 300 nM of 36B4u primer, and 500 nM of36B4d primer, in a final volume of 30 ul per reaction. The thermalcycling profile was 95° C.×10 min. followed by 30 cycles of 95° C. for15 sec. (denaturation) and 56° C. for 1 min. (anneal/extend). No ROX dyewas needed under the second set of conditions.

Similar to amplification of telomere repeats, three 36B4 PCR reactionswere done on each experimental DNA sample. Each 96 well telomere PCRplate contained a row with 2 NTC wells (no genomic DNA), and 10 standardcurve wells containing a serial dilution of a reference DNA to fiveconcentrations, with each concentration in duplicate, ranging from 0.25ng/microliter (final concentration) up to 2 ng/microliter (finalconcentration).

All PCRs were performed on ABI Prism 7700 Sequence Detection System(Applied Biosytems, Inc., Foster City, Calif., USA), a thermal cyclerequipped to excite and read emissions from fluorescent molecules duringeach cycle of the PCR. ABPs SDS version 1.7 software was then used togenerate the standard curve for each plate and to determine the dilutionfactors of standards corresponding to the T and S amounts in eachsample.

In the presence of 35 ng of human DNA, a telomere PCR product wasdetectable by real time quantitative PCR beginning from about 9 cyclesof PCR. Analysis of the product after 25 cycles by electrophoresis onagarose gels and staining with ethidium bromide shows a smear ofproducts beginning from about 76 basepairs, which is equivalent to thesum of the lengths of the telomere specific primers (see FIG. 1), toproducts of about 400 base pairs (FIG. 4). The copy number of the PCR isproportional to the number of sites available for binding of the primerin the first cycle of the PCR. Omitting the genomic DNA results in nodetectable amplification product after 25 cycles for either the telomereor single copy gene primers.

Example 2 Determining the Relative Telomere Length

Mean telomere restriction fragment (TRF) lengths were determined asdescribed by Slagboom et al., Am. J. Hum. Genet. 55: 876-882 (1994),hereby incorporated by reference. Approximately 0.5 ug of purified wholeblood DNA was digested to completion with Hae III restriction enzyme.Digested samples were then mixed with DNA size standards, separated byelectrophresis on agarose gels, and transferred to a nylon membrane. Themembranes were hybridized with ³²P end labeled oligonucleotide,(TTAGGG)₇ (SEQ ID NO: 10) washed to remove non-specifically bound probe,exposed to a phosphor plate for 1 to 5 days, and the plates scanned witha Phosphorlmager (Molecular Dynamics, Inc.). Blots were then stripped ofthe telomere probe, hybridized with radiolabeled probe for the DNA sizestandards, washed, exposed to a phosphor plate, and the plates scanned.The size standard images and telomere smear images were thensuperimposed to locate the positions of the size intervals within thetelomere smears. Mean TRF length was then calculated as mean TRFlength=(ΣOD_(i))/(ΣOD_(i)/L_(i)), where OD_(i) is total radioactivityabove background in interval i, and L_(i) is the average length of i inbasepairs. This entire procedure was performed twice; i.e., the two meanTRF length values determined on each individual were obtained from twoindependent experiments.

To measure the T/S value (telomere to single copy gene ratio), the C_(t)value—the fractional cycle number at which the amplification sample'saccumulating fluorescence crosses a set threshold value that is severalstandard deviations above background fluorescence—was determined forsamples amplified with telomere specific (T) primers and single copygene specific (S) primers. Since the amount of PCR product approximatelydoubles in each cycle of the PCR, the T/S ratio is approximately[2^(Ct(telomeres))/2^(Ct(single copy gene))]⁻¹=2^(−ΔCt). The averageΔC_(t) was −9.05 (see FIG. 2). That is, PCR of a single copy generequired about 9 more cycles than PCR of telomeres to produce equivalentfluorescent signal as measured by real time PCR. The standard deviationwas 1.48%.

The relative T/S ratio, which is the T/S of one sample relative to theT/S of another sample, is expressed as 2^(−(ΔCt1−ΔCt2))=2^(−ΔCt). Thisformula allows calculation of the relative T/S ratio of each sample. DNAsamples of 21 unrelated patients were amplified and quantitated by realtime quantitative PCR (see Experiment 2). Comparison of the relative T/Sratio calculated from PCR correlated well with the mean TRF lengthsdetermined by Southern hybridization (see FIG. 3). The y intercept isabout 3.6 kbp, which is approximately the mean length of thesubtelomeric region between the restriction enzyme recognition sites andthe beginning of the telomeric hexamer repeats (Hultdin, M. NucleicAcids. Res. 26: 3651-3656 (1998)). Moreover, the observed averagetelomere length in whole blood as measured by relative T/S ratio variesover a 2.5 range among unrelated age and sex matched adults. This rangeof variability is in excellent agreement with other studies on the rangeof variation of TRF lengths in age matched adults if the averagesubtelomeric length of 3.4 kbp is subtracted from each reported mean TRFlength (Hultdin, M. Nucleic Acids Res. 3651-3656 (1998); Vaziri, H. etal., Am. J. Hum. Genet. 52: 661-667 (1993)).

1. A method of diagnosing cancer comprising: amplifying repetitive unitsin a repetitive region of a human target nucleic acid comprising: a)contacting a target nucleic acid comprising substantially complementaryfirst and second strands with a first and a second primer, wherein saidfirst primer hybridizes to at least one repetitive unit of said firststrand and said second primer hybridizes to at least one repetitive unitof said second strand, wherein said hybridized primers are capable ofprimer extension when hybridized to their respective strands, andwherein at least one nucleotide of said first primer produces aninternal base pair mismatch between said first primer and a nucleotideof said repetitive unit when said first primer is hybridized to at leastone repetitive unit of said first strand, wherein said first primer alsoproduces a mismatch with the 3′ terminal nucleotide of said secondprimer when first and second primers hybridize to each other, wherein atleast one nucleotide of said second primer produces a an internal basepair mismatch between said second primer and a nucleotide of saidrepetitive unit when said second primer is hybridized to at least onerepetitive unit of said second strand, wherein said second primer alsoproduces a mismatch with the 3′ terminal nucleotide of said first primerwhen first and second primers hybridize to each other; b) amplifying thetarget nucleic acid by the polymerase chain reaction; c) determining thenumber of repetitive units present in the target nucleic acid andcomparing the number of repetitive units to a control; wherein adecrease in the number of repeats indicates the presence of cancer.
 2. Amethod of diagnosing cell senescence comprising: amplifying repetitiveunits in a repetitive region of a human target nucleic acid comprising:a) contacting a target nucleic acid comprising substantiallycomplementary first and second strands with a first and a second primer,wherein said first primer hybridizes to at least one repetitive unit ofsaid first strand and said second primer hybridizes to at least onerepetitive unit of said second strand, wherein said hybridized primersare capable of primer extension when hybridized to their respectivestrands, and wherein at least one nucleotide of said first primerproduces an internal base pair mismatch between said first primer and anucleotide of said repetitive unit when said first primer is hybridizedto at least one repetitive unit of said first strand, wherein said firstprimer also produces a mismatch with the 3′ terminal nucleotide of saidsecond primer when first and second primers hybridize to each other,wherein at least one nucleotide of said second primer produces a aninternal base pair mismatch between said second primer and a nucleotideof said repetitive unit when said second primer is hybridized to atleast one repetitive unit of said second strand, wherein said secondprimer also produces a mismatch with the 3′ terminal nucleotide of saidfirst primer when first and second primers hybridize to each other; b)amplifying the target nucleic acid by the polymerase chain reaction; c)determining the number of repetitive units present in the target nucleicacid and comparing the number of repetitive units to a control; whereina decrease in the number of repeats indicates cell senescence.
 3. Themethod of claim 1, wherein nucleotides residues of said second primerproduce internal base pair mismatches with nucleotides at the identicalnucleotide positions of each repetitive unit of said second strand. 4.The method of claim 1, wherein said first and second primers furthercomprise 5′ terminal sequences which do not hybridize to said repetitiveunits.
 5. The method of claim 1, wherein at least two of the mismatchesin the complex formed by hybridization of said first and said secondprimer are on adjacent nucleotide positions of said repetitive unit. 6.The method of claim 1, wherein said mismatches are on nonadjacentnucleotide positions of said repetitive unit.
 7. The method of claim 1,wherein said repetitive unit comprises hexanucleotide repeats.
 8. Themethod of claim 1, wherein said repetitive unit comprisespentanucleotide repeats.
 9. The method of claim 1, wherein saidrepetitive unit comprise tetranucleotide repeats.
 10. The method ofclaim 1, wherein said repetitive units comprise telomere repetitiveunits.
 11. The method of claim 10, wherein said first primer comprisesSEQ ID NO: 1 and said second primer comprises SEQ ID NO:
 2. 12. Themethod of claim 10, wherein said first primer comprises SEQ ID NO: 8 andsaid second primer comprises SEQ ID NO:
 9. 13. The method of claim 2,wherein nucleotides residues of said second primer produce internal basepair mismatches with nucleotides at the identical nucleotide positionsof each repetitive unit of said second strand.
 14. The method of claim2, wherein said first and second primers further comprise 5′ terminalsequences which do not hybridize to said repetitive units.
 15. Themethod of claim 2, wherein at least two of the mismatches in the complexformed by hybridization of said first and said second primer are onadjacent nucleotide positions of said repetitive unit.
 16. The method ofclaim 2, wherein said mismatches are on nonadjacent nucleotide positionsof said repetitive unit.
 17. The method of claim 2, wherein saidrepetitive unit comprises hexanucleotide repeats.
 18. The method ofclaim 2, wherein said repetitive unit comprises pentanucleotide repeats.19. The method of claim 2, wherein said repetitive unit comprisetetranucleotide repeats.
 20. The method of claim 2, wherein saidrepetitive units comprise telomere repetitive units.
 21. The method ofclaim 20, wherein said first primer comprises SEQ ID NO: 1 and saidsecond primer comprises SEQ ID NO:
 2. 22. The method of claim 20,wherein said first primer comprises SEQ ID NO: 8 and said second primercomprises SEQ ID NO:
 9. 23. A composition for amplifying a targetnucleic acid comprising substantially A composition for amplifying atarget nucleic acid comprising substantially complementary first andsecond target strands, said composition comprising a first and secondprimer, wherein said first primer hybridizes to said first strand andsaid second primer hybridizes to said second strand, wherein saidprimers are capable of primer extension when hybridized to theirrespective strands, and wherein at least one nucleotide residue of saidfirst primer is altered to produce a mismatch between said alteredresidue and the 3′ terminal nucleotide residue of said second primerwhen first and second primers hybridize to each other.
 24. A compositionaccording to claim 23, wherein at least one nucleotide residue of saidsecond primer is altered to produce a mismatch between said alteredresidue on said second primer and the 3′ terminal nucleotide residue ofsaid first primer when first and second primers hybridize to each other.25. A composition for amplifying repetitive units in a repetitive regionof a target nucleic acid comprising substantially complementary firstand second target strands, said composition comprising a first andsecond primer, wherein said first primer hybridizes to at least onerepetitive unit of said first strand and said second primer hybridizesto at least one repetitive unit of said second strand, wherein saidprimers are capable of primer extension when hybridized to theirrespective strands, and wherein at least one nucleotide residue of saidfirst primer is altered to produce a mismatch between said alteredresidue and a nucleotide residue of at least one repetitive unit of saidfirst strand, wherein said altered residue also produces a mismatch withthe 3′ terminal nucleotide residue of said second primer when first andsecond primers hybridize to each other.
 26. A composition for amplifyingrepetitive units in a repetitive region according to claim 25, whereinat least one nucleotide residue of said second primer is altered toproduce a mismatch between said altered residue on said second primerand a nucleotide residue of at least one repetitive unit of said secondstrand, wherein said altered residue on said second primer also producesa mismatch with the 3′ terminal nucleotide of said first primer whenfirst and second primers hybridize to each other.
 27. A composition foramplifying repetitive units in a repetitive region of a target nucleicacid comprising substantially complementary first and second targetstrands, said composition comprising a first and second primer, whereinsaid first primer hybridizes to repetitive units of said first strandand said second primer hybridizes to repetitive units of said secondstrand, wherein said primers are capable of primer extension whenhybridized to their respective strands, and wherein nucleotide residuesof said first primer are altered to produce mismatches between saidaltered residues and nucleotide residues at the identical nucleotideposition of each repetitive unit of said first strand, wherein saidaltered residues also produce a mismatch with the 3′ terminal nucleotideresidue of said second primer when first and second primers hybridize toeach other.
 28. A composition for amplifying repetitive units in arepetitive region according to claim 27, wherein nucleotide residues ofsaid second primer are altered to produce mismatches between saidaltered residues of said second primer and nucleotide residues at theidentical nucleotide position of each repetitive unit of said secondstrand, wherein said altered residues of said second primer also producea mismatch with the 3′ terminal nucleotide residue of said first primerwhen first and second primers hybridize to each other.
 29. A compositionaccording to claim 23, wherein said first and second primers furthercomprise 5′ terminal sequences which do not hybridize to said repetitiveunits.
 30. A composition according to claim 25, wherein said first andsecond primers further comprise 5′ terminal sequences which do nothybridize to said repetitive units.
 31. A composition according to claim27, wherein said first and second primers further comprise 5′ terminalsequences which do not hybridize to said repetitive units.
 32. Acomposition for amplifying repetitive units in a repetitive regionaccording to claim 25, wherein said repetitive units comprise telomererepetitive units.
 33. A composition for amplifying repetitive units in arepetitive region according to claim 27, wherein said repetitive unitscomprise telomere repetitive units.
 34. A composition for amplifyingsaid telomere repetitive units according to claim 33, wherein said firstprimer comprises SEQ ID NO: 1 and said second primer comprises SEQ IDNO:
 2. 35. A composition for amplifying said telomeric repetitive unitsaccording to claim 33, wherein said first primer comprises SEQ ID NO: 8and said second primer comprises SEQ ID NO: 9.